Conversion of liver stem and progenitor cells to pancreatic functional cells

ABSTRACT

The subject invention a method for converting liver stem/progenitor cells to a pancreatic functional cell by transfecting said liver cells with a pancreatic development gene and/or by culturing with pancreatic differentiation factors. The resulting cells produce and secrete insulin protein in response to glucose stimulation.

BACKGROUND

Cell Transplantation as a Cure or Treatment for Diabetes.

Type I diabetes is a chronic metabolic disease caused by selectiveautoimmune destruction of insulin-producing islet β-cells. Clinicalmanagement of diabetes costs ˜$100 billion annually in this country. Theinsulin insufficiency and hyperglycemia of type I diabetes, in the longrun, lead to serious secondary complications. Regular insulinreplacement therapy that is being used to control daily glucosefluctuations, however, does not maintain glucose levels near-normalrange at all times to prevent/reduce clinical complications (The DCCTResearch Group (1991) N. Eng. J. Med. 329:977).

To cure or treat type I diabetes (both in terms of achieving insulinindependence and reducing the incidence of secondary complications), itis essential to restore islet β-cells in the patients either as wholepancreas or islet transplantation. Only about 3,000 cadaver pancreatabecome available in the US each year while ˜35,000 new cases of type Idiabetes are diagnosed during the same period of time (Hering, G. J. etal. (1999) Graft 2:12-27). Thus, there is an urgent need to developalternate sources of functional cells of pancreatic lineage, includingislets and/or insulin-producing cells. The only conceptual optionavailable to circumvent the severe shortage of pancreatic tissue fortransplantation is to develop functional cells of pancreatic lineage(e.g., islets or insulin-producing cells) in vitro from stem cells.

One source of transplantable islets is pancreas-derived islet producingstem cells (IPSCs) (Ramiya, V. K. et al. (2000) Nature Med.6(3):278-282; and PCT/US00/26469, filed Sep. 27, 2000). However,additional/alternative methods of generating pancreatic lineage cellsshould be investigated to increase the chances of success in attempts tocure or treat type I diabetes. Liver stem/progenitor cells offer afeasible source for conversion into pancreatic lineage cells. There aremany advantages of using liver stem cells: a) liver has the immensepotential to regenerate following partial hepactectomy (for instance,the mass and function of the partially hepatectomized liver can betotally restored in about a week, even if ⅔ of liver is resected(Higgins, G. F. et al. (1931) Arc. Pathol. 12:186-202; Grisham, J. W.(1962) Cancer Res. 22:842-849; and Bucher, N. (1963) Int. Rev. Cytol.15:245-300)), and therefore, liver provides a more easily accessiblesource of stem cells for autologous transplantation; and b) the surfacephenotype of liver stem cells have already been established and hence itis easier to purify them from the organ (see Table 1). Also liver stemcells share surface hematopoietic stem cell markers like CD34, Thy1.1,stem cell factor(SCF)/c-kit, Flt-3 ligand/flt-3 (Yin, L. et al. (2001)Proc. Am. Assoc. Canc. Res. 42:354; Yin, L. et al. (2001) FASEB J.Late-Breaking Abstracts:49 (LB267); Fujio, K. et al. (1996) Exp. CellRes. 224:243-50; Blakolmer, K. et al. (1995) Hepatology 21(6):1510-16;Omori, N. et al. (1997) Hepatology 26(3):720-27; Omori, M. et al. (1997)Am. J. Pathol. 150(4): 1179-87; Lemmer, E. R. et al. (1998) J. Hepatol.29:450-454; Petersen, B. E. et al. (1998) Hepatology 27(2):433-445; andBaumann, U. et al. (1999) Hepatology 30(1):112-117), which can be usedfor cell sorting along with other known liver stem cell markers. TABLE 1Markers of development, differentiation and cell lineage specificationof liver epithelial cells (Adapted from Grisham et al. (1991) in StemCells, C. S. Potter (ed.), Academic Press, San Diego, CA, pp. 233-82,with modifications). Bile duct Markers Hepatoblasts Oval cellsHepatocytes cells CK19 + + − + CK14 + + − − Albumin + +/− + − AFP + + −− GGT + + − + OV6 + + − + OV1 + + − + BD1 − − − + HES6 − − + − OC.2 + +− + OC.3 + + − + OC.10 + + − + H.1 − − + − H.4 − − + −

The following sections describe the current status of liver andpancreatic stem cells, their relationship during embryonic developmentand transdifferentiation within these organs.

Development of Liver and Liver Stem Cells.

In the embryo, liver buds from epithelial cells of the ventral foregutin the region that is in contact with the precardiac mesoderm, atapproximately 8.5 to 9 days of development in the mouse. The cells ofthis region proliferate to form the liver diverticulum. At about 9.5days of gestation, cells of the liver diverticulum begin to migrate intothe surrounding septum transversum. At this stage, the cells aredesignated as hepatoblasts, to indicate that these cells have beendetermined along the hepatic epithelial cell lineage. The hepatoblasthas bipotential capability, and gives rise to both hepatocytes and bileduct cells (Houssaint, E. (1980) Cell Differ. 9:269-279). In general,when the liver is injured, the mature hepatocytes proliferate to restorethe mass and function of the liver, and the liver stem cells are notinvolved (Kelly, D. E. et al. (1984) in Bailey's Textbook of MicroscopicAnatomy, 18^(th) ed., Williams and Wilkins, Baltimore, pp. 590-616).However, when the injury is too severe and/or the proliferation ofhepatocytes is inhibited by chemicals such as 2-N-acetylaminofluorene(2AAF) and phenobarbital, the liver stem cell compartment is activated.Liver stem cells in the adult liver have been extensively studied mainlyin the animal liver injury models, such as 2AAF/partial hepatectomy (PH)(Golding, M. et al. (1995) Hepatology 22(4):1243-1253), 2AAF/allylalcohol (AA) and phenobarbitaycocaine leading to periportal liver injury(Yavokovsky, L. et al. (1995) Hepatology 21(6):1702-12; Petersen, B. etal. (1998) Hepatology 27(4):1030-1038; Yin, L. et al. (1999) J.Hepatology 31:497-507; and Rosenberg, D. et al. (2000) Hepatology31(4):948-955), and 2AAF/CCl₄ inducing pericentral liver injury(Petersen et al. (1998)). Irrespective of the injury site, the ovalshaped liver stem cells always originate in the portal area of canals ofHering (Wilson, J. et al. (1958) J. Pathol. Bacteriol. 76:441-449).These liver progenitor cells in adult liver can differentiate into bothhepatocytes and bile duct cells (Stenberg, P. et al. (1991)Carcinogenesis 12:225-231; and Dabeva, J. et al. (1993) Am. J. Pathology143:1606-1620). Most recently, several lines of evidence from bothanimals and humans strongly suggest that hematopoietic stem cells arethe extrahepatic source of liver stem cells (Petersen, B. et al. (1999)Science 284:1168-70; Theise, N. et al. (2000) Hepatology 31(1):235-40;Theise, N. et al. (2000) Hepatology 32(1):11-16; and Alison, M. et al.(2000) Nature 406:257). Epithelial cell lines with stem-like propertieshave been established from mouse liver diverticulum (Rogler, L. (1997)Am. J. Pathol. 150(2):591-602), injured rat liver (Yin, L. et al.(2001A) PAACR 42:354; Yin, L. et al. (2001B) FASEB J. Late-BreakingAbstracts:49 (LB267); Yin, L. et al. (2002) Hepatology 35(2):315-324),and normal rat (Tso, M-S. et al. (1984) Exp. Cell. Res. 154:38-52; andTso, M-S. (1988) Lab. Invest. 58:636-642), porcine (Kano, J. et al.(2000) Am. J. Pathol. 156(6):2033-2043), and human liver (Crosby, H. etal. (2001) Gastroenterology 120(2):534-544). These cells can be inducedto differentiate into hepatocytes and/or bile duct cells in vitro(Rogler, L. (1977); Yin, L. et al. (2001 A); Yin, L. et al. (2001B);Yin, L. et al. (2002); Crosby, H. et al. (2001); and Coleman, W. et al.(1993) Am. J. Pathol. 142:1373-82) and in vivo upon transplantation(Coleman, W. et al. (1993); and Grisham, J. et al. (1993) Proc. Soc.Exp. Biol. Med. 204:270-79).

The signaling molecules that elicit embryonic induction of the liverfrom the mammalian gut endoderm are not fully understood. Fibroblastgrowth factors (FGFs) 1, 2, and 8 expressed in the cardiac mesoderm arereported to be essential for the initial hepatogenesis (Jung, J. et al.(1999) Science 284:1998-2003). Oncostatin M (OSM), an interleukin-6family cytokine, in combination with glucocorticoid, induces maturationof hepatocytes in embryonic liver, which in turn terminate embryonichematopoiesis. Livers from mice deficient for gp130, an OSM receptorsubunit, display defects in maturation of hepatocytes (Kamiya, A. et al.(1999) EMBO J. 18(8):2127-36; and Kinoshita, T. et al. (1999) PNAS96:7265-70). Differentiated hepatocytes are characterized by theexpression of a unique combination of liver-enriched (but notliver-unique) transcription factors of HNF1, HNF3, HNF4, and C/EBPfamilies (Johnson, P. (1990) Cell. Growth Differ. 1:47-51; Lai, E. etal. (1991) Trends Biochem. Sci. 16:427-30; DeSimone, V. et al. (1992)Biochem. Biophys. Acta 1132:119-126; and Crabtree, G. et al. (1992) inTranscriptional Regulation, S. S. McKnight and K. R. Yamamato (eds.)Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 1063-1102).

Development of Pancreas and Pancreatic Stem Cells.

During embryonic development, the pancreas derives from two separateoutgrowths of dorsal and ventral foregut endoderm to form dorsal andventral buds. These buds then fuse to form the definitive pancreas(Houssaint, E. (1980); Spooner, B. et al. (1970) J. Cell Biol.47:235-46; Rutter, W. et al. (1980) Monogr. Pathol. 21:30-38; Guaidi, R.et al. (1996) Genes Dev. 10:1670-82; Zaret, K. (2000) Mech. Dev.92:83-88; Edlund, H. (1998) Diabetes 47:1817-1823; St-Onge, L. et al.(1999) Curr. Opin. Gene Dev. 9:295-300; and Slack, J. (1995)121:1569-80). During embryogenesis, islet development within thepancreas appears to be initiated from undifferentiated precursor cellsassociated primarily with the pancreatic ductal epithelium (Pictet, R.et al. (1992) in Handbook of Physiology, Steiner, D. and Frienkel, N.(eds.) Williams and Wilkins, Baltimore, Md., pp. 25-66). This ductalepithelium rapidly proliferates, and then subsequently differentiatesinto the various islet-associated cell populations (Teitelman, G. et al.(1993) Development 118:1031-39; and Beattie, G. et al. (1994) J. Clin.Endo. Met. 78:1232-1240). In the adult pancreas, the islet cell growthcan occur through two different pathways: either by growth of new isletsby differentiation of ductal epithelium (neogenesis), or by replicationof preexisting β-cells. Neogenesis has been induced experimentally bydietary treatment with soybean trypsin inhibitors (Weaver, C. et al.(1985) Diabetologia 28:781-785), high level of interferon-γ (Gu, D.(1993) Dev. 118:33-46), partial pancreatectomy (Bonner-Weir, S. et al.(1993) Diabetes 42:1715-1720), wrapping of the head of the pancreas incellophane (Rosenberg, L. et al. (1992) Adv. Exp. Med. Biol.321:95-104), and by specific growth factors (Otonkonski, T. et al.(1994) Diabetes 43:947-952). Thus, it is generally accepted that allendocrine cell types of the pancreatic islets arise from the same ductalepithelial stem cell through sequential differentiation (Gu, D. (1993);Rosenberg, L. (1992); and Hellerstrom, D. (1984) Diabetologia26:393-400). Pancreatic stem cells have been isolated from adultpancreatic ductal preparations, and have been shown to differentiate (tosome degree) into insulin-producing cells in vitro (Ramiya, V. et al.(2000); Cornelius, J. et al. (1997) Horm. Metab. Res. 29:271-277; andBonner-Weir, S. et al. (2000) PNAS 97(14):7999-8004), which upontransplantation, were able to reverse diabetes in non-obese diabetic(NOD) mice (Ramiya, V. et al. (2000)).

During embryonic development, there are differences in the specificationof the dorsal and ventral pancreatic rudiments. The dorsalpre-pancreatic endoderm remains closely associated with the notochordduring early developmental stages. Signals derived from overlayingnotochord, such as activin and FGF-2, promote dorsal pancreasdevelopment by repressing endodermal expression of sonic hedgehog (Shh)(Hebrok M. et al. (2000) Dev. 127:4905-13; Kim, S. et al. (1997) Dev.124:4243-52; and Li, H. et al. (1999) Nat. Genet. 23:67-70). Generationof a dorsal pancreas in response to these signaling events also requiresthe expression of a number of transcription factors. For example, mouse‘knockout’ studies have shown that formation of the dorsal pancreas isdependent on Isl1 and Hlxb9, and that subsequent differentiationrequires Pdx1 (Li, H. et al. (1999); Harrison, K. et al. (1999) Nat.Genet. 23:71-75; Ahlgren, U. et al. (1997) Nature 385:257-60). Themechanisms regulating the onset of ventral pancreas development are notfully defined. The control of ventral pancreatic development woulddiffer from that of dorsal pancreas because the notochord does notextend as far as ventral endoderm, and by default, the ventral endodermdoes not express Shh. Moreover, ventral pancreatic development is normalin Isl1 −/− and Hlxb9 −/− mice (Deutsch, G. et al. (2001) Development128:871-881; and Duncan, S. (2001) Nature Genetics 27:355-356). Pdx1 isrequired at an earlier stage in pancreas development (Jonsson, J. et al.(1994) Nature 371:606-609; Ahlgren, U. et al. (1996) Development122:1409-1416; Stoffers, D. et al. (1997) Nat. Genet. 15:106-110; andOffield, M. et al. (1996) Development 122:983-995). Mice and humanslacking Pdx1 are apancreatic (Jonsson, J. et al. (1994); Ahlgren, U. etal. (1996); Stoffers, D. et al. (1997); and Offield, M. et al. (1996)).However, there seems to be other genes that act upstream of Pdx1expression for the initial commitment of the gut endoderm to apancreatic fate. Accordingly, the evagination of the epithelium and theinitial commitment of dorsal and ventral pancreatic buds still takeplace in Pdx1 mutant mice, and insulin- and glucagon-positive cellsstill differentiate (Ahlgren, U. et al. (1996); and Offield, M. et al.(1996)). Later, Pdx1 and Hlxb9 expression in the pancreas becomerestricted to the insulin-producing β-cells (Li, H. et al. (1999);Harrison, K. et al. (1999); and Jonsson, J. et al. (1994)). Pdx1 isrequired for maintaining the hormone-producing phenotype of the β-cellby regulating the expression of a variety of endocrine genes, includinginsulin, GLUT2, glucokinase, and prohormone convertases (PC) 1, 2, and 3(Ahlgren, U. et al. (1998) Genes Dev. 12:1763-68; Hart, A. et al. (2000)Nature 408:864-68; and Baeza, N. et al. (2001) Diabetes 50, Sup. 1:S36).The Pdx1 gene activation may be regulated by HNF3β (Zaret, K. (1996)Annu. Rev. Physiol. 58:231-251) and NeuroD/β2 (Sharma, T. et al. (1997)Mol. Cell Biol. 17:2598-2404). Several homeodomain and basichelix-loop-helix (bHLH) transcription factors like ngn3, Isl1, Nkx2.2,Nkx6. 1, Pax4, Pax6, and NeuroD/β2, have been shown to play an importantrole in the control of pancreatic endocrine cell differentiation(Edlund, H. (1998); St-Onge, L. et al. (1999); Sander, M. et al. (1997)J. Mol. Med. 75:327-340; Madsen, O. et al. (1997) Horm. Metab. Res.29(6):265-270; and Gradwohl, G. et al. (2000) PNAS 97(4):1607-11). Ofthese genes, ngn3 has been reported to be critical for the developmentof all four endocrine cell lineages of the pancreas (Gradwohl, G. et al.(2000)). Pax4 appear to selectively control the development ofinsulin-producing β-cells and somatostatin-producing δ-cells(Sosa-Pineda, B. et al. (1997) Nature 386:399-402). Nkx6.1 has a highlyrestricted β-cell expression in the adult rat (Madsen, O. et al.(1997)). Disruption of Nkx6.1 in mice leads to loss of β-cell precursorsand blocks β-cell neogenesis (Sander, M. et al. (2000) Dev.127(24):5533-5540). Thus, it is essential to screen for these factorsfollowing differentiation procedures to determine the extent ofdifferentiation.

Various growth factors, hormones, vitamins and chemicals, such ashepatocyte growth factor (HGF), glucagon-like peptide-1 (GLP-1),exendin-4, activin-A, β-cellulin, dexamethasone, nicotinamide, andsodium butyrate, have been shown to be effective in β-celldifferentiation in vitro. HGF (Mashima, H. et al. (1996) Endocrinol.137:3969-76), GLP-1 (Zhou, J. et al. (1999) Diabetes 48:2358-2366),exendin-4 (Zhou, J. et al. (1999)), dexamethasone, β-cellulin andactivin-A (Mashima, H. et al. (1996) J. Clin. Invest. 97(7):1647-54)differentiate acinar cells into insulin-secreting cells. GLP-1 increaseslevels of β-cell cAMP and insulin gene transcription and stimulatesglucose-dependent insulin release (Grucker, D. et al. (1987) PNAS84:3434-3438). Administration of GLP-1 for 10 days to neonatal diabeticrats following partial pancreatectomy stimulated expansion of β-cellmass via induction of islet proliferation and neogenesis (Xu, G. et al.(2000) Diabetes 48:2270-76). GLP-1 also increases Pdx1 gene expressionand binding capacity (Buteau, J. et al. (1999) Diabetes 49:1156-1164).Exendin-4 is a potent structural analog of GLP-1, and has a longercirculating half-life. It binds to GLP-1 receptor on islets with similaraffinity to GLP-1, but increases cAMP levels 3-fold higher than GLP-1 atequimolar concentrations, malking it a more effective agent for use inchronic animal studies (Garcia-Ocana, A. et al. (2001) JCE & M86:984-988). Dexamethasone and sodium butyrate might promote β-celldifferentiation as evidenced by increased insulin/DNA contents inporcine pancreatic islet-like cell clusters (Korsgren, O. et al. (1993)Ups. J. Med. Sci. 98(1):39-52). In pancreas cell line, RIN-m5F, sodiumbutyrate increases 2-fold both hexokinase and glucokinase activities, aswell as, the glucokinase gene expression. Nicotinamide is a poly(ADP-ribose) synthetase inhibitor known to differentiate and increaseβ-cell mass in cultured human fetal pancreatic cells and mouse IPSCs(Ramiya, V. et al. (2000); and Otonkoski, T. et al. (1993) J. Clin.Invest. 92:1459-66) and prevents the development of diabetes in druginduced diabetic animal models as well as in the NOD mice (Uchigata, Y.et al. (1983) Diabetes 32:316-18; and Yamada, K. et al. (1982) Diabetes31:749-753).

Manipulation of Pancreatic Stem and Liver Stem Cell Differentiation.

In embryonic development, the liver and ventral pancreas both originatefrom the same location in the ventral foregut (Houssaint, E. (1980);Rutter, W. (1980); Guaidi, R. et al. (1996); Zaret, K. (2000); Deutsch,G. et al. (2001); and Zaret, K. (1996)). Therefore, it is possible, fromthe developmental point of view, that epithelial cells in these twoorgans may share common stem cells. A new study shows that a bipotentialcell population exists in the embryonic endoderm that gives rise to boththe liver and the pancreas. The decision by these cells to adopt eithera pancreatic or hepatic cell fate is determined by their proximity tothe developing heart (Deutsch, G. et al. (2001)). The defaultdevelopmental program of the ventral endoderm is to become ventralpancreas. Several lines of evidence have attested to the ability ofpancreatic stem cells to differentiate into liver cell. For instance,copper depletion and repletion result in the atrophy of exocrinepancreas and the appearance of oval cells within the pancreatic ductswhich then differentiate into hepatocytes within the pancreas (Rao, M.et al. (1986) Cell Differ. 18:109-117; Rao, M. et al. (1988) Biochem.Biophys. Res. Commun. 156:131-136; and Reddy, J. et al. (1991) Dig. Dis.Sci. 36(4):502-509). Oval cells with immunophenotype identical tohepatic stem cells were also found in human pancreas with acutepancreatitis, chronic pancreatitis, and pesidioblastosis (Mikami, Y. etal. (1998) Hepatology 28(4), Pt. 4:417A). The pancreatic hepatocytesrespond to the carcinogens in a fashion similar to liver hepatocytes(Rao, M. et al. (1991) Am. J. Pathol. 139(5):1111-1117). Followingtransplantation into the liver, pancreatic oval cells isolated fromcopper-deficient rat pancreas can differentiate into mature hepatocyteswith structural integration in the hepatic parenchyma and expression ofbiochemical functions unique to the hepatocytes (Dabeva, J. et al.(1997) PNAS 94:7356-61). Most recently, Wang and coworkers demonstratedthe existence of undifferentiated progenitors of hepatocytes in thepancreas of normal adult mouse (Wang, X. et al. (2001) Am. J. Pathol.158:571-79). Pancreatic cells can also be converted into hepatocytes invitro by treatment with dexamethasone (Shen, C-N. et al. (2000) NatureCell Biol. 2:879-887). The initial events involve activation of thetranscriptional factor C/EBP-β. Transfection of cells with C/EBP-βbrings about hepatic differentiation. Therefore, C/EBP-β is suggested asa key component that distinguishes the liver and pancreatic programs ofdifferentiation. The consistent development of pancreatic hepatocytes intransgenic mice overexpressing KGF driven by insulin promoter indicatesthe involvement of KGF in the transdifferentiation process (Krakowski,M. et al. (1999) am. J. Pathol. 154(3):683-91). Although not specific toendocrine cells, there is also a report on the ability of liver togenerate pancreatic epithelial cells (Rao, M. et al. (1986) Histochem.Cytochem. 34:197-201; and Bisgaard, H. et al. (1991) J. Cell Physiol.147(2):333-343). Further, liver transduced with recombinant-adenoviruscarrying gene encoding Pdx1 can produce functional insulin andameliorates streptozotocin-induced diabetes in mice; however, Pdx1 isreported to not transdifferentiate liver hepatocytes to insulinproducing cells in vitro, and no evidence is provided that mouse liverstem or progenitor cells are transfected in vivo (or in vitro) with thePdx1 construct (Ferber, S. et al. (2000) Nature Med. 6(5):568-571).Finally, while sharing of transcription factors such as Isl1, ngn3,NeuroD/β2, Pax4, pax6, and Nkx2.2, between endocrine and neuronaldifferentiation pathways has been established (Ahlgren, U. et al.(1997); Sander, M. et al. (1997); Sosa-Pineda, B. et al. (1997); Pfaff,S. et al. (1996) Cell 84:309-320; Lee, J. et al. (1995) Science268:836-844; Naya, F. et al. (1997) Genes Dev. 11:2323-2334; Miyata, T.et al. (1999) Genes Dev. 13:1647-52; St-Onge, L. et al. (1997) Nature387:406-409; Ericson, J. et al. (1997) J. Cell 90:169-180; Sussel, L. etal. (1998) Dev. 125:2213-2221; Briscoe, J. et al. (1999) Nature398:622-627), there is no clear information on the sharing oftranscription factors between liver and pancreas.

SUMMARY OF THE INVENTION

The subject invention comprises methods of culturing liverstem/progenitor cells with combinations of hormones, growth factors,vitamins and chemicals to convert the liver stem or progenitor cells topancreatic functional cells. It further comprises transfection methodsfor conversion of liver stem or progenitor cells to pancreaticfunctional cells.

Thus, the invention provides a method for converting a liverstem/progenitor cell to the pancreatic functional cell by transfectingthe liver stem/progenitor cell with a pancreatic development gene.Alternatively, the liver stem/progenitor cell may be cultured underconditions that convert the cell to the pancreatic functional cell.Further, conversion can be achieved by both transfection and cultureconditions, effected simultaneously or sequentially in either order.

The liver stem/progenitor cell can be a hepatoblast or a liver ovalcell. It is preferred that the liver stem/progenitor cell express atleast one hematopoietic marker and/or at least one liver oval orhepatoblast cell marker. The hematopoietic markers include CD34, Thy1.1and CD45. The liver hepatoblast or oval cell markers include α-fetalprotein, albumin, cytokeratin 14(CK14), c-kit, OC.2, OC.3, OC.10, OV1and OV6.

The pancreatic development gene is any gene that is capable ofconverting liver stem/progenitor cells to pancreatic functional cells,and includes Pdx1, Hlxb9, Isl1, ngn3, Nkx2.2, Pax6, NeuroD/β2, Nkx6.1and Pax4. Preferably, the pancreatic development gene is Pdx-1.

Culture conditions that convert liver stem/progenitor cells to thepancreatic functional cells comprise basal medium plus the added factorsof hormones, growth factors, vitamins and chemicals or any combinationthereof that induce differentiation into pancreatic cells. Such hormonesinclude dexamethasone, glucagon-like peptide-1 (GLP-1), and exendin-4;growth factors include gastrin, interferon-γ (IFNγ), hepatocyte growthfactor (HGF), epidermal growth factor (EGF), β-cellulin, activin-A,keratinocyte growth factor (KGF), fibroblast growth factor (FGF),transforming growth factor-α (TGF-α), transforming growth factor-β(TGF-β), nerve growth factor (NGF), insulin-like growth factors (IGFs),islet neogenesis associated protein (INGAP), and vascular endothelialgrowth factor (VEGF); vitamins include nicotinamide and retinoic acid;and chemicals include sodium butyrate.

According to this method, the liver stem/progenitor cell that isconverted can express any combination of a number of pancreaticmessenger RNAs, including insulin I (InsI), insulin II (InsII),glucagon, somatostatin, pancreatic polypeptide (PP), amylase, elastase,glucose transporter 2 (GLUT2), glucokinase, PC1, PC2, PC3,carboxypeptidase E (CPE), Pdx1, Hlxb9, Isl1, ngn3, Nkx2.2, Pax6,NeuroD/β2, Nkx6.1 and Pax4. Likewise, the converted cell may express anycombination of a number of pancreatic proteins including InsI, InsII,glucagon, somatostatin, PP, amylase, elastase, GLUT2, glucokinase, PC1,PC2, PC3, CPE, Pdx1, Hlxb9, Isl1, ngn3, Nkx2.2, Pax6, NeuroD/β2, Nkx6.1and Pax4.

Preferably, the converted liver stem/progenitor cell differentiates intothe pancreatic endocrine pathway. Such converted cells can be culturedto produce endocrine hormones (e.g., insulin, glucagon and somatostatinfrom β, α and δ cells).

The method of conversion via transfection with a pancreatic developmentgene or via culture conditions may result in pancreatic cells atdifferent stages of differentiation, including islet producing stemcells (IPSCs), islet progenitor cells (IPCs) and islet-like structuresor IPC-derived islets (IdIs), or cellular components thereof (α, β, δand/or PP cells). Transdifferentiation may also result in a cell thatmanifests expression patterns of a pancreatic cell (e.g., insulinproduction), and that may also retain characteristics of the liverstem/pancreatic cell (e.g., liver stem or progenitor markers). Liverstem/progenitor cell markers include hematopoietic markers and liveroval or hepatoblast cell markers.

All references cited herein are incorporated in their entirety byreference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 sets forth the characterization of five liver epithelial celllines derived from allyl alcohol-injured rat liver. The meaning ofsymbols is: − negative, +/− weakly positive, + positive, ++ stronglypositive.

FIG. 2 illustrates the bipotentiality of liver epithelial line 3(8)#21to differentiate into hepatocyte-like and bile duct-like cells. A:3(8)#21 cultured without feeder are positive for mature hepatocytemarker H4. B: on day 6, 3(8)#21 cultured without feeder are positive formature hepatocyte marker CYPIAII on day 12. C: 3(8)#21 cultured withoutfeeder but with bFGF; more H4 positive cells observed on day 6. D:3(8)#21 cells cultured on matrigel without feeder form ductularstructure on day 4. E: 3(8)#21 cells cultured on matrigel without feederexpress strongly mature bile duct cell marker BD1 on day 13. Themagnification is 400× for panels A, B, C, and E. The magnification is200× for panel D (Yin, L. et al. (2001A); Yin et al. (2001B); and Yin,L. et al. (2002)).

FIG. 3 shows the expression of pancreatic development markers in fiveliver stem/progenitor cell lines.

FIG. 4 illustrates the expression of insulin II and amylase in the liverprogenitor lines after transfection with the Pdx1 gene.

DETAILED DESCRIPTION

To facilitate a further understanding of the invention, the followingdefinitions are provided.

“Islet producing stem cell” (IPSC) refers to those stem cells that arisefrom or among pancreatic ductal epithelium in vitro and in vivo. Methodsfor obtaining and maintaining IPSCs are described in detail inPCT/US00/26469, filed Sep. 27, 2000, which incorporated herein in itsentirety by reference.

“Islet progenitor cell” (IPC) refers to pancreatic progenitor cells thatarise from IPSCs cultured in vitro using methods described herein and inPCT/US00/26469.

“IPC-derived islet” (IdI) refers to the islet-like structures that arisefrom IPCs cultured in vitro using methods described herein and inPCT/US00/26469.

“Liver stem/progenitor cell” refers to all liver stem and/or progenitorcells, including without limitation, hepatoblasts, oval cells, liverepithelial cells with stem-like properties, and de-differentiatedhepatocytes and bile duct cells. While many liver stem/progenitor lineshave been reported in the literature (Williams, G. et al. (1971) Exp.Cell Res. 69:106-112; Williams, G et al. (1973) 29:293-303; Grisham, J.(1980) Ann. N.Y. Acad. Sci. 349:128-137; Tsao, M-S. et al. (1984) Exp.Cell Res. 154:38-52; Coleman, W. et al. (1997) Am. J. Pathol.151:353-359; Coleman, W. et al. (1993) Am. J. Pathol. 142:1372-82;McCullough, K. et al. (1994) Cancer Res. 54:3668-71; Amicone, L. et al.(1997) EMBO J. 16:495-503; Spagnoli, F. et al. (1998) J. Cell Biol.143:1101-1112; Sell, S. et al. (1982) Hepatol. 2:77-86; Shinozuka, H. etal. (1978) Cancer Res. 38:1092-98; McMahon, J. et al. (1986) Cancer Res.46:4665-71; Brill, S. et al. (1999) Digest. Dis. Sci. 44:364-71; andRogler, L. (1977) Am. J. Pathol. 150:591-602), preferably, the liverstem/progenitor cells used in the subject methods are obtained fromliver injury models without the involvement of carcinogens, as describedfor example in Yin, L. et al. (2001A), Yin, L. et al. (2001B) and Yin,L. et al. (2002). It is also preferred that the liver stem/progenitorcells express one or more of the liver oval or hepatoblast cell markers(α-fetal protein, albumin, cytokeratin 14 (CK14), c-kit, OC.2, OC.3,OC.10, OV1 and OV6), and/or one or more of the hematopoietic stemmarkers (CD34, Thy1.1 and CD45).

“Pancreatic endocrine lineage” refers to commitment to development intopancreatic endocrine cells.

“Pancreatic lineage” refers to commitment to development into pancreaticcells including endocrine, exocrine and/or duct cells.

“Pancreatic functional cells” refers to cells of the pancreatic lineageor cells that have been transdifferentiated or converted according tomethods described herein, and which express mRNA or proteins that arecharacteristic of and specific to a pancreatic cell (e.g., insulin), andwhich may also retain characteristics of the liver stem/pancreatic cell(i.e., liver stem or progenitor markers). The pancreatic functional cellpreferably is a glucose-responsive, insulin producing cell. Itpreferably produces and secretes insulin protein in response to glucosestimulation. The response is preferably within the normal range ofinsulin response for the mammalian species of interest. Such normalranges are known in the art or are readily determinable.

“Transfection” refers to any method known in the art by which a fragmentor construct of nucleic acid containing a coding sequence may beintroduced into a target cell (here, a liver stem/progenitor cell)resulting in the expression of the coding sequence in the target cell.Included within the fragment or construct are the requisite promoter andregulatory sequences for expression in the target cell.

Thus, the subject invention comprises a method of converting a liverstem/progenitor cell to a pancreatic functional cell, by transfectingthe liver stem/progenitor cell with a pancreatic development gene,and/or by culturing said liver stem/progenitor cell in a mediumcomprising factors that induce differentiation into the pancreaticfunctional cell. The resulting pancreatic functional cell can be a cellof the pancreatic endocrine lineage, or can be a cell having anexpression pattern that is intermediate between the liverstem/progenitor cell and cells of pancreatic lineage. The term “cells ofthe pancreatic lineage” means islet producing stem cells (IPSCs), isletprogenitor cells (IPCs), islet-like structures or IPC-derived islets(IdIs), or naturally derived pancreatic endocrine cells (e.g., α, βand/or δ cells, or duct cells). Additionally, cells having anintermediate expression pattern are those that produce and secreteinsulin protein in response to glucose stimulation, and which mayexpress a marker of the liver stem/progenitor cell.

The liver stem/progenitor cells can be hepatoblasts and/or liver ovalcells. The liver stem/progenitor cell expresses at least onehematopoietic marker and/or at least one liver oval or hepatoblast cellmarker. The hematopoietic markers are CD34, Thy1.1 and/or CD45. Thehepatoblast or oval cell markers α-fetal protein, albumin, cytokeratin14 (CK14), c-kit, OC.2, OC.3, OC.10, OV1 and/or OV6.

In the transfection embodiment, the pancreatic development gene can bePdx1, Hlxb9, Isl1, ngn3, Nlkx2.2, Pax6, NeuroD/β2, Nkx6.1 and/or Pax4.Preferably, the pancreatic development is Pdx-1.

In the culture transdifferentiation embodiment, liver stem/progenitorcells are cultured under methods known in the art in a standard mediumplus factors. The factors include dexamethasone, glucagon-like peptide-1(GLP-1), exendin-4, gastrin, interferon-γ (IFNγ), hepatocyte growthfactor (HGF), epidermal growth factor (EGF), β-cellulin, activin-A,keratinocyte growth factor (KGF), fibroblast growth factor (FGF),transforming growth factor-α (TGF-α), transforming growth factor-β(TGF-β), nerve growth factor (NGF), insulin-like growth factors (IGFs),islet neogenesis associated protein (INGAP), vascular endothelial growthfactor (VEGF), nicotinamide, retinoic acid, sodium butyrate or anycombination thereof.

The converted cell can express any of a number of pancreatic messagesincluding insulin I (InsI), insulin II (InsII), glucagon, somatostatin,pancreatic polypeptide (PP), amylase, elastase, glucose transporter 2(GLUT2), glucokinase, PC1, PC2, PC3, carboxypeptidase E (CPE), Pdx1,Hlxb9, Isl1, ngn3, Nkx2.2, Pax6, NeuroD/β2, Nkx6.1 and/or Pax4.Accordingly, the converted cell can express pancreatic proteinsincluding InsI, InsII, glucagon, somatostatin, PP, amylase, elastase,GLUT2, glucokinase, PC1, PC2, PC3, CPE, Pdx1, Hlxb9, Isl1, ngn3, Nkx2.2,Pax6, NeuroD/β2, Nkx6.1 and Pax4. It is preferred, however, that theconverted cell produce and secrete insulin protein in response toglucose stimulation. The response is preferably within normal range forthe mammalian cell of interest.

The subject invention also comprises a pancreatic functional cellproduced by the methods described herein, wherein the pancreaticfunctional cell has an expression pattern that is intermediate betweenthat of the liver stem/progenitor cell and cells of pancreatic lineage.In one embodiment, the pancreatic functional cell expresses Pdx1,amylase and insulin II.

The invention further comprises a method for producing an endocrinehormone comprising converting the liver stem/progenitor cells topancreatic functional cells as described herein, culturing saidpancreatic functional cells using methods known in the art andrecovering endocrine hormone from the cell culture using methods knownin the art.

EXAMPLES Example 1 Liver Stem/Progenitor Cells

Liver epithelial cell lines with liver stem cell properties weredeveloped from allyl alcohol (AA)-injured adult rat liver as describedin Yin, L. et al. (2001 A); Yin, L. et al. (2001B); and Yin, L. et al.(2002). AA induces periportal liver injury, which is a liver injurymodel without the involvement of hepatocarcinogens (Peterson B. E. etal. (1998) Hepatology 27(4):1030-38). Five cell lines, named 1(1)#3,1(1)#6, 1(3)#3, 2(11) and 3(8)#21, were chosen to investigate theirpotential of differentiation to pancreatic lineage cells. These 5 lineshave been well characterized by Western blot, Northern blot,immunocytochemistry and histochemistry for various liver developmental,cell lineage markers and hematopoietic stem cell markers. The resultsare summarized in FIG. 1. Pictures of the immunocytochemistry results ofthe line 3(8)#21 are also presented in FIG. 1. Interestingly, almost allof the lines express hematopoietic stem cell markers CD34, Thy1.1, andCD45 indicating their possible relationship to hematopoietic stem cells.They also express liver progenitor cell genes such as α-fetal protein(AFP), albumin, cytokeratin 14 (CK14) and c-kit. They do not express Itocell marker Desmin or Kupffer cell/macrophage markers, ED1 and ED2(results not shown). These cells can be maintained in theirundifferentiated status without the expression of maturehepatocyte-specific genes such as glucose-6-phosphatase (G-6-Pase),dipeptidyl peptidase IV (DPPIV), and cytochrome P450 (CYP450), andwithout showing the expression of mature bile duct cell-specific geneCK19 (results not shown). All 5 lines are diploid by flow cytometry.Induction of differentiation carried out in line 3(8)#21 shows thathepatocyte phenotype can be induced by long-term culture without STOfibroblast feeder layer (FIG. 2 A, B). Basic FGF is able to augment thedifferentiation (FIG. 2 C). Culturing the cells on matrigel inducesliver bile duct phenotype (FIG. 2 D, E). These results suggestbipotentiality of line 3(8)#21 to differentiate into hepatocytes orliver biliary cells.

Example 2 Expression of Pancreatic Developmental and Cell LineageSpecific Genes in Liver Progenitor Cell Lines

These five (untreated) liver progenitor cell lines have been analyzedfor the expression of selected pancreatic endocrine markers includinginsulin I and insulin II, pancreatic exocrine marker amylase, GLUT2(glucose transporter), and some of the transcription factors that arecritically involved in the development of pancreas such as Pdx1, Isl1,NeuroD/β2, Nkx6.1 and Pax4. The expression of these genes was determinedby RT-PCR, and confirmed by Southern blot. The data is presented in FIG.3. Rat pancreatic tissue expresses most of the markers tested includinginsulin I, insulin II, amylase, GLUT2, Pdx1, Isl1, and Nkx6.1 but notNeuroD/β2 and Pax4 (FIG. 3; lane 1). Gamma-irradiated STO feeder cellsdo not express any of these markers (FIG. 3; lane 2). Liver progenitorcell lines 1 (1)#3 (FIG. 3; lane 3) and 3(8)#21 (FIG. 3; lane 7) expressalmost all the pancreatic transcription factors tested and even insulinI and II, but they do not express detectable levels of amylase, Pdx1 andGLUT2 (FIG. 3; lanes 3 & 7). Cell line 1 (1)#6 is positive for INSI andII and NeuroD/β2 (FIG. 3 lane 4). Cell line 2(11) are positive forNeuroD/β2, Nkx6.1 and Pax4 but negative for all other markers (FIG. 3;lane 6). Cell line 1(3)#3 is only positive for NeuroD/β2 (FIG. 3; lane5). These data indicate that at least 2 of the 5 liver progenitor lines(1(1)#3 and 3(8)21) tested express their “preparedness” to enter intopancreatic pathway even before any treatment with islet-differentiatingfactors.

Additionally, and as a positive control, the pancreas-determiningtranscription factor, Pdx1 was transfected into each of these liverprogenitor lines with the aim of directing the liver stem cells intopancreatic differentiation pathway. As shown in FIG. 4, the introductionof Pdx1 gene triggers the expression of amylase gene (FIG. 4; lanes3,5,7,9,11), which is not expressed in the non-transfected parentallines (FIG. 4; lanes 2,4,6,8,10). Interestingly, cell lines 1(1)#3, and3(8)#21 which express insulin II exhibit reduction of insulin IIexpression following transfection with Pdx1 gene (FIG. 4; lanes2,3,10,11) while induction of insulin II was seen in cell lines that didnot express the gene prior to transfection (FIG. 4; lanes 4,5,6,7).

Example 3 Characterization of Expression in Liver Stem/progenitor CellsUnder Different Experimental Conditions so as to Determine TheirDifferentiation Potential

Liver progenitor lines described herein are studied for expression ofgenes controlling the pancreatic development at different stages (Pdx1,Hlxb9, Isl1, ngn3, Nkx2.2, Pax6, NeuroD/β2, Nkx6.1, and Pax4), endocrinecell lineage markers (insulin I, insulin II, glucagon, somatostatin, andPP), exocrine markers (amylase and elastase), and the genes associatedwith insulin sensing, synthesis, process and secretion (GLUT-2,glucokinase, PC1, PC2, PC3 and carboxypeptidase E (CPE)). Each cell lineis evaluated for expression under untreated and treated conditions.Treated cell lines are those that are grown under culture conditionsknown to enhance differentiation of pancreatic stem or progenitor cells,and/or are transfected with pancreatic development genes.

RT-PCR, Southern blot, immunocytochemistry, and Western blot techniquesare used to determine the gene expression both at mRNA expression (allgenes) and at protein levels (e.g. Pdx1 and hormones). Normal pancreatictissue, primary hepatocytes, and STO feeder cells serve as controls. Thetreated cell lines are characterized and compared to untreated lines.Expression of liver stem cell markers (AFP, albumin, CK14, c-kit, OV6,OV1) and hematopoietic stem/progenitor cell markers (CD34, Thy1.1, CD45)are also analyzed in the treated lines to see if their liver stem cellphenotypes are lost after treatment.

Plasmids such as plasmid pBKCMV/Stf1 (Pdx1) carrying Pdx1 gene and Neogene (a gift from Dr. Dutta, Hoffmann-La Roche, Inc. Nutley, N.J.), areused to transfect liver cell lines. The Pdx1 transfected cells can beused as a positive control for differentiation into insulin-producingcells.

RT-PCR/Southern Blot

DNA-free RNA is extracted by using StrataPrep™ Total RNA Miniprep Kit(Stratagene, La Jolla, Calif.) or RNAqueous™-4PCR Kit (Ambion, Austin,Tex.) by following the manufacture's protocol. RT-PCR is carried outfollowing methods known in the art. The oligonucleotides used asamplimers for PCR are listed in Table 1. PCR cycle is at 95° C. for 3min followed by 94° C. for 45 sec, corresponding optimized annealingtemperature for each primer pair is 45 sec, 72° C. for 1 min (34cycles), and 72° C. for 10 min. PCR products are run in 1.5% Seakemagarose gel in TBE buffer using a BioRad/RAC300 power supply at 100 voltfor 80 min. The gel is incubated in 1% ethidium bromide solution in TBEbuffer for 15 to 30 min, and then viewed using UV light. Image isphotographed and processed using AlphaImage™2200 Documentation &Analysis system (Alpha Innotech Corporation, San Leandro, Calif.).Digoxigenin-labeling of an Oligo probe for Southern blotting is carriedout by using Dig Oligonucleotide Tailing Kit (Roche MolecularBiochemicals, Indianapolis, Ind.) by following the manufacture'sprotocol. As a corroborative technique, Southern blotting is carried outfollowing the PCR reaction using the standard protocol. TABLE 3Oligonucleotides used as amplimers for PCR. Gene Sense primer Antisenseprimer Size GenBank accession Pdx1 ACATCTCCCCATACGAAGTGCCGGAGCTGGCAGTGATGCTCAACT 364 U04833(R) Isl1 ACGTCTGATTTCCCTATGTGTTGGCCGCTCTAAGGTGTACCACATCGA 276 S69329(R) Hlxb9 CAGCACCCGGCGCTCTCCTAGAACTGGTGCTCCAGCTCCAGCAGC 250 NM-005515(Hu) Ngn3CCTGCAGCTCAGCTGAACTTGGCGA GCTCAGTGCCAACTCGCTCTT 485 AJ133776(Hu) Nkx2.2CCGAGAAAGGTATGGAGGTGAC CTGGGCGTTGTACTGCATGTGCTG 187 X81408(Ha) Nkx6.1ATGGGAAGAGAAAACACACCAGAC GAACGAGGAGGACGACGACGATTA 280 AF004431(R) Pax4TGGCTTTCTGTCCTTCTGTGAGG TCCAAGACTCCTGTGCGGTAGTAG 214 AF053100(R) Pax6AAGAGTGGCGACTCCAGAAGTTG CCTGAAGCAAGGATACAGGTGTGGT 545 U69644(R)NeuroD/β2 AGCCATGAATGCAGAGGAGGAC ACACTCTGCAAAGGTTTGTCC 400 AF107728(R)Insulin I ATGGCCCTGTGGATGCGCTT CTGGAGAACTACTGCAACT 331 J00747(R) InsulinII ATGGCCCTGTGGATCCGCTT GTGACCTTCAGACCTTGGCA 243 V01243(R) GlucagonGTGGCTGGATTGTTTGTAATGCTG GTTGATGAACACCAAGAGGAACCG 236 NM-012707(R) PPTGAACAGAGGGCTCAATACGAAAC GATTTGTAGCCTCCCTTCTGTCT 214 M18207(R) AmylaseGCCTTGGTGGGAAAGATATC TCCCAAGGAAGCAGACCTTT 510 V01225(R) ElastaseGTGAGCAGCCAGATGACTTTCC CCTGGATGAACAATGTCATTG 573 NM-012552(R) GLUT-2TTAGCAACTGGGTCTGCAAT CATGAGTGTAGGACTACACC 343 J03145(R) GlucokinaseAGAGTGATGCTGGTCAAAGTGGGA ATGATTGTGGGCACTGGCTGCAAT 440 J04218(R) G3PDHGCCATCACTGCCACCCAGAAG GTCCACCACCCTGTTGCTGCA 440 M32599(R)Ha, hamster; Hu, human; R, rat.

Immunocytochemistry

An avidin-biotin method adapted from Biogenex (San Ramon, Calif.) isfollowed. Cells are either cytospun onto Fisher Brand superfrost plusslides (Fisher Scientific, Pittsburgh, Pa.) using a cytocentrifuge(Cytopro™, Wescor, Inc. Logan, Utah) or grown onto 8 well glass slides(ICN Costa Mesa, Calif.) placed in tissue culture plates (Falcon®,Becton Dickinson, Franklin Lakes, N.J.), and fixed in 0.5%glutaraldehyde for 1 hr at room temperature. For intracellular staining,the cells are permeabilized using 0.2% Triton-X. Blocking and antigenretrieval (when necessary) is done prior to primary antibody staining ofthe cells. Secondary antibodies are conjugated to biotin which is linkedto alkaline phosphatase or horseradish peroxidase; and streptavidin,which binds to the biotin, is linked to alkaline phosphatase orperoxidase. Antibodies are then visualized using 3,3′-diaminobenzidine(DAB), 3-amino-9-ethylcarbzole (AEC), Fast Red, or5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBI).This system also can be used for double staining which can visualizemulti-antigen expression.

Western Blot

Western blot is also used for the gene translation study. Cells arelysed with lysis buffer (for 8.0 ml: 3.8 ml of dH₂O, 1 ml of 0.5 MTris-HCl (pH 6.8), 0.8 ml of glycerol, 1.6 ml of 10% (w/v) SDS, and 0.4ml of β-mercaptoethanol, and 0.4 ml of 0.5% TABLE 4 Factors and theirfinal concentrations in the medium Basal Medium (BM) Components DMEM(Dulbecco's Minimum Essential Medium, Gibco-BRL) 0.1 mM2-Mercaptoethanol 15% FBS (HyClone) 1x ITS (insulin, transferrin andselenium) (Gibco-BRL) Penicillin 100 IU/ml and streptomycin 100 μg/mlFungizone ® 1 μg/ml (Gibco-BRL) 200 mM L-glutamine 1X non-essentialamino acids (Gibco-BRL) Factors to be added to BM Dexamethasone 10⁻⁷ MGLP-1 (glucagon-like peptide) 10 nM Exendin-4 (10 nM) Gastrin (10 nM)IFNγ (interferon-γ) 0.1-2 ng/ml HGF (hepatocyte growth factor) 20 ng/mlEGF (epidermal growth factor) 10 ng/ml Betacellulin 5 nM Activin A 1 nMKGF (keratinocyte growth factor) 5-10 ng/ml FGF (fibroblast growthfactor) 10 ng/ml TGF-alpha (transforming growth factor) 10-15 ng/mlTGF-beta NGF (nerve growth factor) 25-50 ng/ml IGFs (insulin-like growthfactor) 10 ng/ml INGAP (islet neogenesis associated protein) 125 ng/mlVEGF (vascular endothelial growth factor) 10-20 ng/ml Nicotinamide 10 mMRetinoic Acid 1 ng/ml Sodium butyrate 2.5 mMbromophenol blue). Tissues are homogenized on ice in homogenizationbuffer (20 mM Tris, 137 mM NaCl, 10% glycerol, 1 mM Na₃VO₄, 1 u/mlaprotinin, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), pH8.0), centrifuged at 9,000 g for 20 min at 4° C., and the supernatantcollected. The protein concentration is determined using Coomassie PlusProtein Assay Reagent (PIERCE, Rockford, Ill.). Samples are then run onseparating gel at appropriate concentration at 100 volts, 4 watts, and50 mAs for 2 hr. Gel is then transferred using Mini Trans-Blot Cell(Bio-Rad, Hercules, Calif.) to nitrocellulose membrane (Bio-Rad) intransfer buffer (25 mM Tris, 192 mM glycine and 20% v/v methanol, pH8.3) at 30 volts, 2 watts, and 50 mAs overnight. The membrane is thenblotted in respective primary antibody at 4° C. overnight. Thereafter,the membrane is washed and incubated with the corresponding secondaryantibody linked with alkaline phosphatase for 1 hr at room temperature,and developed in carbonate buffer (0.1 M NaHCO₃, 1 mM MgCl₂, pH 9.8)containing 60 μl of nitro blue tetrazolium (NBT) solution (dissolve 50mg of NBT in 0.7 ml of N,N-Dimethylformamide (DMF) with 0.3 ml dH₂O),and 60 μl of 5-bromo4-chloro-3-indolyl phosphate (BCIP) solution(dissolve 50 mg of BCIP in 1 ml of 100% DMF) until appropriate colorobtained.

Differentiation of Liver Stem Cells

Liver stem cells are cultured in basal medium (BM) containingcombinations of hormones (dexamethasone, GLP-1, exendin-4), growthfactors (gastrin, interferon-γ (IGFγ), HGF, EGF, β-cellulin, activin-A,KGF, FGF, TGF-α & -β, NGF, IGFs, INGAP, and VEGF), vitamins(nicotinamide, and retinoic acid), and/or chemicals (sodium butyrate),at concentrations listed in Table 4. The concentrations set forth inTable 4 may be varied by 1-3 orders of magnitude so as to optimize theireffectiveness. The foregoing hormones, growth factors, vitamins andchemicals are reported in the literature or in PCT Application No.PCT/US02/09881, filed Mar. 29, 2002, to be involved in pancreas/β-celldevelopment. Expression of liver stem cells markers and hematopoieticstem cell markers are also observed in the treated lines to determinewhether their liver stem cell phenotypes are lost after treatment.

Transfection and Selection

FuGENE 6 transfection reagent (Roche Molecular Biochemicals,Indianapolis, Ind.) is used to transfect pBKCMV/Stf1 (Pdx1) carryingPdx1 gene and Neogene into liver stem cell lines using manufacturer'sprotocol. Mock transfection and vector alone transfection are also doneat the same time. Three days after gene transfection, the cells arecultured in the culture medium containing G418 1 mg/ml. The resistantclones are grown out in about ten days. The selection is carried out for2 to 4 weeks. Thereafter, the cells are cultured in the culture mediumcontaining 0.3 mg/ml of G418.

Analogous transfections can be carried out with plasmids containingother pancreatic development genes.

Example 4 Determination of the Functional Capability of Rat LiverStem/progenitor Cell-derived Insulin-producing Cells (LSDIPCs)

The cell lines of Example 3 that are found to produce insulin (LSDIPCs)are further evaluated for their glucose responsiveness. The cells aretested for both extra- and intracellular insulin production. Where acell line demonstrates glucose responsive insulin production, then thesubstrate phosphorylation pattern can be determined following glucosestimulation. Freshly isolated rat islet cells serve as a positivecontrol. Observation of substrate phosphorylation pattern reveals theearly signaling events involved in the induction of insulin production.

Glucose Induced Insulin-stimulation Assay

Differentiated LSDIPCs are seeded at a concentration of 2×10⁵ cells perwell in 24 well plates with 1 ml of medium containing 5.5 mM glucose for24 hr to rest. Cells are washed with Krebs-Ringer buffer (KRB) and arestimulated with 1 ml of culture medium with or without glucose (0, 5.5,11 and 17.5 mM glucose) for 3-18 hrs. The cell free supernatant iscollected and stored at −70° C. until use. The cells are then treatedwith lysis buffer to determine insulin content using MercodiaUltrasensitive Rat Insulin ELISA Enzyme immunoassay kit (Mercodia,Uppsala, Sweden). This insulin kit is used to measure both secreted andintracellular insulin using BioRad's Benchmark plate reader (490 nm).The insulin values are normalized to total DNA concentrations (extractedusing Trizol™, Gibco) of cells.

ELISAs for Hormone Detection

As mentioned above, secreted and intracellular insulin are measuredusing Mercodia Ultrasensitive Rat Insulin ELISA Enzyme immunoassay kit(Mercodia, Uppsala, Sweden) following the manufacturer's protocol.Similarly, a glucagon assay is carried out using methods known in theart or adaptations thereof.

Substrate Phosphorylation Assay

Differentiated LSDIPCs are homogenized in extraction buffer (20 mmol/lK₂HPO4, pH 7.5, 5 mmol/l DTT, 1 mmol/l EDTA, and 110 mmol/l KCL)following stimulation with 17.5 mM glucose for 0, 5, 15 and 30 min. Thehomogenate is used to separate protein on 10% SDS-PAGE (BioRad) and thephosphorylated protein substrates are detected usinganti-phosphotyrosine antibody (Pharmingen, San Diego, Calif.) in Westernblot technique.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

1. A method of converting a liver stem/progenitor cell to a pancreatic functional cell, wherein said pancreatic functional cell produces and secretes insulin in response to glucose stimulation, said method comprising: transfecting said liver stem/progenitor cell with a pancreatic development gene, culturing said liver stem/progenitor cell in a medium comprising factors that induce differentiation into the pancreatic functional cell, or both, whereby said transfected cell is converted to the pancreatic functional cell.
 2. The method of claim 1, wherein the pancreatic functional cell is a cell of the pancreatic endocrine lineage.
 3. The method of claim 2, wherein said converted cell is selected from the group consisting of islet producing stem cells (IPSCs), islet progenitor cells (IPCs) and islet-like structures or IPC-derived islets (IdIs).
 4. The method of claim 1, wherein the pancreatic functional cell further expresses a marker of the liver stem/progenitor cell.
 5. The method of claim 1, wherein said liver stem/progenitor cell is selected from the group consisting of hepatoblasts and liver oval cells.
 6. The method of claim 1, wherein said liver stem/progenitor cell expresses at least one marker selected from the group consisting of hematopoietic markers and liver oval or hepatoblast cell markers.
 7. The method of claim 6, wherein said hematopoietic markers are selected from the group consisting of CD34, Thy1.1 and CD45.
 8. The method of claim 6, wherein said liver hepatoblast or oval cell markers are selected from the group consisting of α-fetal protein, albumin, cytokeratin 14 (CK14), c-kit, OC.2, OC.3, OC.10, OV1 and OV6.
 9. The method of claim 1, wherein said pancreatic development gene is selected from the group consisting of Pdx1, Hlxb9, Isl1, ngn3, Nkx2.2 Pax6, NeuroD/β2, Nkx6.1 and Pax4.
 10. The method of claim 9, wherein said pancreatic development gene is Pdx-1.
 11. The method of claim 1, wherein said factors are selected from the group consisting of dexamethasone, glucagon-like peptide-1 (GLP-1), exendin-4, gastrin, interferon-γ (IFNγ), hepatocyte growth factor (HGF), epidermal growth factor (EGF), β-cellulin, activin-A, keratinocyte growth factor (KGF), fibroblast growth factor (FGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), nerve growth factor (NGF), insulin-like growth factors (IGFs), islet neogenesis associated protein (INGAP), vascular endothelial growth factor (VEGF), nicotinamide, retinoic acid, sodium butyrate and any combination thereof.
 12. The method of claim 1, wherein said converted cell expresses a pancreatic message selected from the group consisting of insulin I (InsI), insulin II (InsII), glucagon, somatostatin, pancreatic polypeptide (PP), amylase, elastase, glucose transporter 2 (GLUT2), glucokinase, PC1, PC2, PC3, carboxypeptidase E (CPE), Pdx1, Hlxb9, Isl1, ngn3, Nkx2.2, Pax6, NeuroD/β2, Nkx6.1 and Pax4.
 13. The method of claim 1, wherein said converted cell expresses a pancreatic protein selected from the group consisting of InsI, InsII, glucagon, somatostatin, PP, amylase, elastase, GLUT2, glucokinase, PC1, PC2, PC3, CPE, Pdx1, Hlxb9, Isl1, ngn3, Nkx2.2, Pax6, NeuroD/β2, Nkx6.1 and Pax4.
 14. A pancreatic functional cell produced by the method of claim
 1. 15. The pancreatic functional cell of claim 14, wherein the cell further expresses Pdx1, amylase and insulin II.
 16. A method for producing an endocrine hormone comprising converting liver stem/progenitor cells according to the method of claim 1, and further comprising: culturing said converted cells; and recovering endocrine hormone from said cell culture.
 17. A liver stem/progenitor cell that has been transfected with a pancreatic development gene.
 18. The method of claim 17, wherein said liver stem/progenitor cell is selected from the group consisting of hepatoblasts and liver oval cells.
 19. The liver stem/progenitor cell of claim 17 wherein the pancreatic development gene is selected from the group consisting of Pdx1, Hlxb9, Isl1, ngn3, Nkx2.2, Pax6, NeuroD/β2, Nkx6.1 and Pax4.
 20. The liver stem/progenitor cell of claim 19 wherein the pancreatic development gene is Pdx1.
 21. A liver stem/progenitor cell that has been cultured in a medium comprising factors that induce differentiation into a pancreatic functional cell.
 22. The liver stem/progenitor cell of claim 21 wherein said factors are selected from the group consisting of dexamethasone, glucagon-like peptide-1 (GLP-1), exendin4, gastrin, interferon-γ (IFNγ), hepatocyte growth factor (HGF), epidermal growth factor (EGF), β-cellulin, activin-A, keratinocyte growth factor (KGF), fibroblast growth factor (FGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), nerve growth factor (NGF), insulin-like growth factors (IGFs), islet neogenesis associated protein (INGAP), vascular endothelial growth factor (VEGF), nicotinamide, retinoic acid, sodium butyrate and any combination thereof. 